2. CONTENT
 Definition
 Objective
 Preliminary testing
 Different media
 Sampling
 Methods
Total aerobic microbial count
 Membrane filtration
 Plate count
 Serial dilution

3. DEFINITION
 This test are designed to perform qualitative &
quantitative estimation of the no. of viable aerobic
micro-organisms present or detecting the presence of
designated microbial species in pharmaceutical
product.
 The term ‘growth’ is used to designate the presence
& presumed proliferation of viable micro-organism.
3

4. OBJECTIVE[1]
 Microbial limit tests are designed to estimate the
number of viable aerobic organisms present in
pharmaceutical products and raw materials.
 The microbial limit testing of raw material as well as
finished pharmaceutical products can help to
determine whether the product complies with
requirement of IP.
 The most care must be taken while performing
microbial test so that contamination from outside can
be avoided.
4

5. Preliminary Testing[7]
 The method given herein are invalid unless it is
demonstrated that the specimen to which they
are applied do not themselves inhibit the
multiplication of under the test condition of
micro-organism that can be present .
 Therefore, inoculate diluted specimen of
substance being examined with separate viable
culture of
(1)E.coli
(2)S.aures
(3)S.typhi
(4)Psudomonas aeruginosa
5

6. 1 ml of NLT 10-3 dilutions of 24 hr broth culture
+
buffer solution pH 7.2, fluid soyabean-casein
digest medium or fluid lactose medium
+
test material
inoculate incubate
6

7. Continue . . .
 If the organisms fail to grow in the relevant medium
the procedure should be modified by
(a) increasing the volume of diluents with the quantity
of test material remaining the same, or
(b) incorporating a sufficient quantity of a suitable
inactivating agent in the diluents, or
(c) combining the above modifications so as to permit
growth of the organisms in the media.
 If inhibitory substances are present in the sample,
0.5% of soya lecithin and 4% of polysorbate 20 may
be added to the culture medium.
7

8. Continue . . .
 Alternatively, repeat the test as described using fluid
casein digest-soya lecithin- polysorbate 20 medium to
demonstrate neutralization of preservatives
OR
other antimicrobial agents in the test material.
8

9. Media[1]
 Baird – Parker Agar Medium
 Bismuth Sulphite Agar Medium
 Brilliant Green Agar Medium
 Buffered Sodium Chloride-Peptone Solution pH 7.0
 Casein Soyabean Digest Agar Medium
 Cetrimide Agar Medium
 Desoxycholate-Citrate Agar Medium
 Fluid Casein Digest-Soya Lecithin-Polysorbate 20
Medium
9

10. Continue:
 Fluid Lactose Medium
 Lactose broth Medium
 Levin Eosin-Methylene Blue Agar Medium
 MacConkey Agar Medium (culture of enterobacteria)
 MacConkey Broth Medium
 Mannitol Salt Agar Medium
 Nutrient Broth Medium
 Nutrient Agar Medium
 Pseudomonas Agar Medium for Detection of
Flourescein
10

11. Continue:
 Pseudomonas Agar Medium for Detection of
Pyocyanin
 Sabouraud Dextrose Agar Medium
 Sabouraud Dextrose Agar Medium with Antibiotics
 Selenite F Broth
 Fluid Soyabean-Casein Digest Medium
 Tetrathionate-Bile-Brilliant Green Broth Medium
 Triple Sugar-Iorn Agar Medium
 Urea Broth Medium
 Vogel-Johnson Agar Medium
 Xylose-Lysine-Desoxycholate Agar Medium
(selective media for salmonella)
11

12. Notes[1]
 Where agar is specified in a formula, use agar that has a
moisture content of not more than 15%.
 Where water is called for in a formula, use purified
water.
 The media should be sterilized by heating in an
autoclave at 115°c for 30 minutes.
 In preparing media dissolve the soluble solids in the
water, using heat if necessary, to effect complete
solution an add solutions of hydrochloric acid or sodium
hydroxide in quantities sufficient to yield the required
pH in the medium when it is ready for use. Determine
the pH at 25°c ± 2°c
12

13. Some common ingredients and its use[8]
Agar:
 A solidifying agent which is a complex polysaccharide
derived from marine algae.
 It has no nutritional value in media.
 It is bacteriological inert.
 It is stable at different temperature used for incubation.
Peptones:
 Protein is large, relatively insoluble molecules that a
minority of organism can utilized directly, but a partial
digestion by acid or enzyme reduces protein to shorter
chain of amino acids called peptone. These small, soluble
fragments can be digested by most bacteria.
 It should be stored in a tightly closed container as it is
hygroscopic in nature.
13

14. Continue:
Meat extract:
 It is prepared from fresh meat by hot water extraction.
 It contains water soluble constituents of animal tissue
that is carbohydrates, organic nitrogen compound,
water soluble vitamins and mineral salts.
Yeast extract:
 It is particularly rich in vitamin B.
 It also contains carbohydrates, amino acids, inorganic
salts, growth factors.
14

15. TERMS[8]
 Culture medium: A nutrient material prepared for
growth of micro-organism in a laboratory is called
culture medium.
 Culture: The microbes can grow and multiply in or on a
culture medium are referred to as a culture.
 Nutrient broth: if the complex media is in liquid form, it
is called nutrient broth.
 Nutrient agar: when agar is added to media, it is called
nutrient agar.
15

16. TYPES OF CULTURE MEDIA[8]
 Selective: suppression of unwanted microbes and
encouraging desired microbes.
 Differential: differentiation of colonies of desired
microbes from others.
 Enrichment: similar to selective but designed to
increase numbers of desired microbes to detectable
levels.
16

17. SAMPLING[1]:
 Use 10 ml or 10 g specimens for each of the tests
specified in the individual monograph.
PRECAUTION:
 The microbial limit tests should be carried out under
conditions designed to avoid accidental contamination
during the test.
 The precautions taken to avoid contamination must be
such that, they do not adversely effect any micro
organism that should be revealed in the test.
17

18. 18

19. METHODS[1]:
1. TOTAL AEROBIC MICROBIAL COUNT:
Unit: cfu/ml or gm[3]
 colony-forming unit (CFU or cfu) is a measure of viable
bacterial or fungal numbers. Unlike direct microscopic
counts where all cells, dead and living, are counted, CFU
measures viable cells. For convenience the results are
given as CFU/ml (colony-forming units per milliliter) for
liquids, and CFU/g (colony-forming units per gram) for
solids.
 This technique allows the determination of the number of
CFU per ml in the sample, and thus the degree of
contamination in samples of water, vegetables, soil or
fruits, and in industrial products and equipment.
19

20. Pre-treatment of sample:-
To dissolve or dilute the sample use
 Phosphate buffer (pH 7.2)
 Sodium chloridepeptone buffer solution
 Fluid medium used for the test.
If not specified:-
 Use 10 g or 10 ml of the sample
 Adjust the test fluid to pH 6.8
 Use the test fluid within one hour after
preparation.
20

21. Water soluble products:
10 g or 10 ml of the sample + buffer or fluid medium
mix
make up to 100 ml.
If necessary, adjust the pH to about 7.
21

22. Products insoluble in water (non-fatty) :
Take 10 g or 10 ml of the sample
grind
fine powder + buffer or fluid medium
suspend(use blender or surfactant to disperse )
Make it up to 100 ml
A suitable surface-active agent such as 0.1% w/v of
polysorbate 80 may be added to assist the suspension of
poorly wettable substances.
22

23. Fatty products :
10 g or 10 ml of the sample + buffer or fluid medium
+surfactant (polysorbate 20 or polysorbate 80)
Prepare emulsion and make up to 100 ml.
 If necessary, warm at a temperature not exceeding
40˚c to emulsify the sample.
 Avoid warming for more than 30 minutes.
23

24. A. Membrane filtration method
10 ml or dilution containing 1 gm sample
membrane filter(50 mm in diameter,
pore size NGT 0.45 µm)
Residue
 Wash it with buffered sodium chloride-peptone solution
pH 7.0 [For fatty substances add to the liquid
polysorbate 20 or polysorbate 80.]
 Transfer the filter on media for enumeration
24

25. Membrane filtration assembly and
A sterile screw-capped container[4][6]
25

26. •Count the number of colonies that are formed. Calculate the
number of micro-organisms per gm or per ml of the
preparation being examined.
26

27. Laminar air flow unit[6]
27

28. B. Plate count method[1]
a)Pour -plate method
b)Surface -spread method
a)Pour plate method :-
Take Petri dishes 9 to 10 cm in diameter
1 ml of the pretreated preparation + 15ml (15-20 ml as per U.S.P) of
liquified media at NMT 45°c.
 If necessary, dilute the pretreated preparation .
28

29. b) Surface-spread method:-
 Spread the pretreated preparation on the surface of the
solidified media in a Petri dish of the same diameter .
 Prepare at least two such Petri dishes using the same
dilution and incubate.
 If necessary dilute the pretreated preparation
 For bacteria :- Count the 300 colonies per plate as the
maximum consistent with good evaluation.
 For fungi :- Calculate the results using plates with not
more than 100 colonies. 29

30. 30

31. Plate showing the colony[11] colony counter[12]
31

32. C. Serial Dilution Method (Multiple tube method)
[9]
 Use 12 test tubes: 9 containing 9 ml of soybean-casein
digest medium each and 3 containing 10 ml of the same
medium each for control.
 Prepare dilutions using the 9 tubes.
 First, add 1 ml of the test fluid to each of three test tubes
and mix to make 10- times dilutions.
 Second, add 1 ml of each of the 10-times dilutions to each
of another three test tubes and mix to make 100-times
dilutions.
 Third, add 1 ml of each of the 100-times dilutions to each
of the remaining three test tubes and mix to make 1,000-
times dilutions. 32

33. Continue:
 Incubate all 12 test tubes for at least 5 days at 30 - 35°c.
No microbial growth should be observed for the control
test tubes.
 If the determination of the result is difficult or if the
result is not reliable, take a 0.1ml fluid from each of the 9
test tubes and place it to an agar medium or fluid
medium, incubate all media for 24-72 hours at 30°-35°c,
and check them for the absence or presence of microbial
growth.
 Calculate the most probable number of microorganisms
per ml or gram of the sample
33

34. 2. TESTS FOR SPECIFIED MICRO
ORGANISMS
 As per IP
 Escherichia coli
 Salmonella
 Pseudomonas aeruginosa
 Staphylococcus aureus
 Preparetion of test fluid:-
 Proceed as described under the test for total aerobic
microbial count .
 Using lactose broth or medium which have no
antimicrobial activity in place of buffered sodium
chloride-peptone solution pH 7.0. 34

35. Escherichia coli[3][6] is a Gram negative rod-shaped bacterium that is
commonly found in the lower intestine of warm-blooded organisms
(endotherms). Most E. coli strains are harmless, but some, such as
serotype O157:H7, can cause serious food poisoning in humans, and
are occasionally responsible for product recalls. The harmless strains
are part of the normal flora of the gut, and can benefit their hosts by
producing vitamin K2 and by preventing the establishment of
pathogenic bacteria within the intestine.
35

36. As per IP
Escherichia coli[1]
Take sterile screw-capped container
add sample+50 ml of nutrient broth
shake
allow to stand for 1 hour (4 hours for gelatin)
Shake it again
Loosen the cap
Incubate at 36°c to 38°c for 18 to 24 hours
36

37. Continue:
 Primary test –
 1.0 ml culture to a 5 ml of MacConkey broth. Incubate
at 36°c to 38°c for 48 hours.
 If the contents of the tube shows acid and gas, carry out
secondary test.
 Secondary test –
Add 0.1 ml of the contents to
(a) 5 ml of MacConkey broth, and
(b) 5 ml of peptone water
37

38.  Incubate in a water-bath at 43.5°c to 44.5°c for 24
hours and examine tube
(a) for acid and gas
(b) for indole
 For indole add 0.5 ml of Kovac’s reagent, shake well.
 If a red colour is produced in the reagent layer indole
is present.
 The presence of acid, gas and indole in the secondary
test indicates the presence of Escherichia coli.
38

39. Continue:
 Carry out a control test by repeating the primary and
secondary tests adding 1.0 ml of the enrichment culture
and a volume of broth containing 10 to 50 Escherichia
coli organisms, prepared from a 24 hour culture in
nutrient broth, to 5 ml of MacConkey broth.
 The test is not valid unless the results indicate that
control contains Escherichia coli.
 Kovac's reagent[10]: A reagent used to detect the presence
of indole which is used in identification of bacteria.
 Indole test[10] :The indole test is a biochemical test
performed on bacterial species to determine the ability of
the organism to split indole from the amino acid
tryptophan.
39

40. Salmonella[3][6] is a genus of rod-shaped, Gram-negative,
non-spore forming, predominantly motile enterobacteria.
Flagella which project in all directions (i.e. peritrichous).
They cause illnesses like typhoid fever, paratyphoid fever,
and the food borne illness.
40

41. Salmonella[1]
Take sterile screw-capped container
add
sample+100 ml of nutrient broth
shake
allow to stand for 4 hour
Shake it again
Loosen the cap
incubate at 35°c to 37°c for 24 hours
41

42. Primary Test - Add 1.0 ml of the culture to each of the two
tubes containing
(a) 10 ml of selenite F broth
(b) Tetrathionate bile-brilliant green broth
 Incubate at 36ºc to 38ºc for 48 hours.
 From each of these two cultures, subculture on at least
two of the following four agar media: bismuth sulphite
agar, brillinat green agar, desoxycholate-citrate agar and
xylosetysine desoxycholate agar.
 Incubate the plates at 36ºc to 38ºc for 18 to 24
hours
42

43. Test for salmonella:
43

44. Continue:
 Upon examination, if none of the colonies conforms to
the description given in Table, the sample meets the
requirements of the test for the absence for the genus
Salmonella.
 If any colonies conforming to the description in Table
are produced, carry out the secondary test.
 Secondary test:
 Subculture any colonies showing the characteristics
given in Table in triple sugar- iron agar by first
inoculating the surface of the slope and then making a
stab culture with the same inoculating needle, and at the
same time inoculate a tube of urea broth.
 Incubate at 36ºc to 38ºc for 18 to 24 hours.
44

45. Continue:
 The formation of acid and gas in the stab culture.
 The absence of acidity from the surface growth.
 The absence of a red colour in the urea broth.
Indicates the presence of salmonella
 Carry out the control test by repeating the primary and
secondary test using 1.0 ml of the enrichment culture and
a volume of broth containing 10 to 50 salmonella
organisms, prepared from a 24-hour culture in nutrient
broth.
 The test is not valid unless the results indicate that the
control contains Salmonella.
45

46. Pseudomonas aeruginosa[3]
 It is a gram negative bacterium which can cause disease
in humans and non-human animals. It is found in soil,
water, skin flora, and most man-made environments
throughout the world.
 It uses a wide range of organic material for food; in
animals, the versatility enables the organism to infect
damaged tissues or people with reduced immunity. The
symptoms of such infections are generalized
inflammation and sepsis. If such colonization occur in
critical body organs such as the lungs, the urinary tract,
and kidneys, the results can be fatal.
46

47. Continue[3][6]
 P. aeruginosa strains produce two types of soluble pigments, the
fluorescent pigment pyoverdin and the blue pigment pyocyanin.
The latter is produced abundantly in media of low-iron content
and functions in iron metabolism in the bacterium. Pyocyanin
refers to "blue pus", which is a characteristic of infections caused
by Pseudomonas aeruginosa.
47

48. 1 ml or 1 gm containg sample + 100 ml of fluid
soyabean-casein digest medium
Mix
Incubate at 35ºc to 37ºc for 24 to 48 hours.
 If growth is present, streak a portion of the medium on
the surface of petri dishes of cetrimide agar medium.
 Cover and incubate at 35ºc to 37ºc for 18 to 24 hours.
 If upon examination, none of the plates contains colonies
having the characteristics listed in Table for the media used,
the sample meets the requirement for freedom from
Pseudomonas aeruginosa.
 If any colonies conforming to the description in Table are
produced, carry out the oxidase and pigment tests.
48

49. Tests for Pseudomonas aeruginosa
49

50. Oxidase and pigment tests:
 Streak representative suspect colonies from agar surface
of cetrimide agar on the surfaces of pseudomonas agar
medium for detection of fluorescein and for detection of
pyocyanin contained in Petri dishes.
 Cover and invert the inoculated media and incubate at
33º to 37º for not less than 3 days. Examine the streaked
surfaces under ultra-violet light. Examine the plates to
determine whether colonies conforming to the
description in previous table are present.
50

51. Continue:
 If growth of suspect colonies occurs, place 2 or 3 drops
of a freshly prepared 1% w/v solution of N, N, N1, N1 –
tetramethyl-4-phenylenediamine dihydrochloride on filter
paper and smear with colony; if there is no development
of a pink color, changing to purple, the sample meets the
requirements of the test for the absence of Pseudomonas
aeruginosa.
51

52. Staphylococcus aureus [3][6]
S. aureus are gram positive cocci (in clusters) which can
cause a range of illnesses from minor skin infections, such as
pimples, scalded skin syndrome, to life-threatening diseases
such as pneumonia, meningitis, osteomyelitis, endocarditis,
chest pain, bacteremia, and sepsis. Its incidence is from skin,
soft tissue, respiratory, bone, joint, endovascular to wound
infections.
52

53. Staphylococcus aureus[1]
 Proceed as described under Pseudomonas aeruginosa.
If, upon examination of the incubated plates, none of
them contains colonies having the characteristics listed
in Table for the media used, the sample meets the
requirements for the absence of Staphylococcus aureus.
 If growth occurs, carry out the coagulase test.
53

54. Continue:
54

55. Continue:
 Coagulase test:
 Transfer representative suspect colonies from the agar
surface of any of the media listed in Table to individual
tubes, each containing 0.5 ml of mammalian- preferably
rabbit or horse-plasma with or without additives.
 Incubate in water-bath at 37º examining the tubes at 3
hours and subsequently at suitable intervals up to 24
hours.
 If no coagulation in any degree is observed, the sample
meets the requirements of the test for the absence of
Staphylococcus aureus.
55

56. APPLICATION
 MICROBIOLOGICAL ASSAY OF ANTIBIOTIC
DRUGS
 DISINFECTION EFFICACY TEST OF
DISINFECTANTS AND ANTISEPTICS
 STERILITY TEST OF STERILISED
PHARMACEUTICALS
 TESTS FOR MICROBIAL LIMITS FOR NON-
STERILE PHARMACEUTICAL AND BIOLOGICAL
PRODUCTS
 TESTING OF WATER
56

57. REFERENCES
1. The Indian pharmacopoeia, IP-1996, volume-2, Indian
pharmacopoeia commission, Ghaziabad, India, 1996.
Appendix-9.4,A-110:A-117
2. The United States Pharmacopoeia,USP-25 NF-20,
2002, p 1873-1878
3. http://en.wikipedia.org
4. http://productimage.tradeindia.com/00530916/s/1/Me
mbrane-Filtration-Assembly-PC-Single-Unit-.jpg
5. http://en.wikipedia.org/wiki/Candida_albicans
6. http://www.google.co.in/image
7. http://www.usp.org/pdf/EN/meetings/asMeetingIndia2
009/session2Track2Tirumalai_1.pdf
57

58. Continue:
1. Gerard J. Tortora, Berdell R. Funke, Christine L. Case,
Microbiology An Introduction, Eighth Edition, Pearson
Education, 2005, p 193-199
2. http://ffcr.or.jp/zaidan/FFCRHOME.nsf
3. http://en.wikipedia.org/wiki/Indole_test
4. http://biology.clc.uc.edu/fankhauser/Labs/Microbiolog
y/Yeast_Plate_Count/07_yeast_0.2mL_plate_P720118
1.jpg
5. http://chemicoscientific.com/images/Digital_Colony_C
ounter.jpg
58

59. 59